Compositions and methods for the removal of tattoos

ABSTRACT

The present invention provides a method for removing a tattoo in a region of skin the method comprises administering to a least a portion of the tattoo a composition comprising an effective amount of a bisphosphonate and at least one pharmaceutically acceptable excipient to at least cause fading of the tattoo in said region.

CROSS REFERENCE TO PRIOR APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 15/337,945, filed Oct. 28, 2016, which is a divisional of U.S.patent application Ser. No. 14/914,929, filed Feb. 26, 2016, issued asU.S. Pat. No. 9,801,799 on Oct. 31, 2017, which is a 371 national phaseapplication of PCT/CA2014/000663, filed Aug. 28, 2014, which claims thebenefit of priority to U.S. Provisional Application No. 61/871,929 filedon Aug. 30, 2013, the disclosures of which are all incorporated hereinby reference in their entireties.

FIELD

The present invention generally relates to compositions and their use inthe treatment and removal of body art, particularly, tattoo removal.

BACKGROUND

The statements in this section merely provide background informationrelated to the present disclosure and may not constitute prior art.

Tattoos are a popular method of body art, and there are an estimated20-30 million tattooed individuals in the world. Approximately 50% ofthose who get tattoos regret all or part of their tattoos and want tohave them removed completely, or faded enough to allow for re-tattooingof a new design over the old tattoo. Currently, there are no reliable,safe, consistent, or affordable methods for tattoo removal. Laser-basedtherapies are the standard of care and have showed promise in veryspecific tattoos (i.e. pigment type, size, and patient's skin color),but have several shortcomings that prevent many from seeking thistreatment.

Professional tattoos are created by injecting tattoo inks with a rapidlyreciprocating needle that drives ink particles into the dermis to adepth of 0.6 mm to 2.2 mm. The inks used in tattooing are derived fromexogenous pigments. Pigments in tattoo ink can include iron oxides,chromium oxide, aluminum oxide, titanium oxide, barium sulfate, zincoxide, sodium copper silicate, sodium aluminum silicate, coppercarbonate, dioxazine and carbazole among other known pigmentformulations. Following injection of the ink pigment particles onto aregion of skin, the ink pigment particles reside in the interstitialspace between dermal cells where they form large aggregates of about 100μm to 200 μm until fibroblasts or macrophages engulf the pigmentparticles and internalize the tattoo ink. The size of the ink particleaggregates and the collagen network surrounding the aggregates help keepthe ink pigments within the skin making the tattoo permanent, thus thedifficulty with removal of tattoos.

Tattoo removal depends upon several factors including size of thetattoo, location of the tattoo, the individual's healing process, howthe tattoo was applied and the length of time it has been on the skin.Tattoos naturally fade over time due to sun exposure and the ongoingimmune response to foreign bodies (i.e. ink pigment particles).Laser-based therapies use high-energy light emissions at specificwavelengths to, in essence, accelerate and enhance specificity of thenatural decomposition of tattoo ink by sun exposure. As tattoo inks varyin color and consequently, absorption wavelengths, lasers with differentemission wavelengths are necessary to remove specific pigments,essentially requiring advanced and expensive laser treatments withsophisticated lasers that can emit several wavelengths. The high energylight decomposes the ink residing in residual bodies, allowing thenatural immune response to continue. The drawbacks of laser-basedtherapies for tattoo removal include and are not limited toaffordability, availability, reliability, consistency, associated painand discomfort, hypo- or hyper-pigmentation of the skin, and scarring.Other commonly accepted treatment modalities for tattoo removal includedermabrasion and surgical removal which may further increase the risk ofadverse side effects and potential irreversible markings left in theremoval process.

In addition to the more prevalent body tattoos, cosmetic tattoos or‘permanent make-up’ are also skin problems that many try to remove withlaser, dermabrasion and surgical treatments. In some examples ofcosmetic tattoos, tattooed eyebrows, eyeliner, and lip liner are alsooften sought to be removed.

When working around the eye area with laser or surgical treatment,particular care must be taken to ensure that there is no eye damage,necessitating the use of protective eye shields that sit on the corneafor the removal of permanent eyeliner tattoos. The laser used to removethese pigments can also remove hair (but only temporarily) and this canbe a problem when removing eyeliner, and eyebrow tattoos.

Furthermore, with cosmetic tattoos, it is possible for a shift in colorto occur e.g. a pink lip liner tattoo may become dark green or black.Although, this darker color can still be treated with laser, it may notappear aesthetically pleasing during the period of removal and furtherincreases the inconvenience, time and cost for permanent removal.

IBIS World (Tattoo Removal Practitioners Market Research Report, January2012) values the tattoo removal market at $66 Million and determined a21% annual growth from 2007-2012. To put the out-of-pocket cost inperspective, the average starting cost per square inch of tattoo removalcan range from about $100.00 to about $150.00 (averaging approximately$125 per treatment) and it takes 6-10 treatments depending on the sizeand ink colors to obliterate most tattoos. It is therefore reasonable tobelieve, that the cost of removing a tattoo with laser therapies is fargreater than the original cost of the tattoo. It follows that analternative therapy would be desirable both from a customer andeconomical perspective.

For at least the reasons provided above, there is a need to providedermatological procedures for safe, reliable and economical methods forthe removal or fading of permanent tattoos.

SUMMARY

The present technology provides novel compositions for the removal andfading of tattoos applied to the skin of a mammalian subject.

In another aspect the present technology provides a method for removinga tattoo in a region of skin the method comprising: administering to aleast a portion of the tattoo a composition comprising an effectiveamount of a bisphosphonate and at least one pharmaceutically acceptableexcipient to at least cause fading of the tattoo in said region.

In one aspect, the composition is an intradermal administrablecomposition, a topically administrable compositions or a transdermaldevice. In a related aspect, the composition provides bisphosphonateparticles suitable for administration in a region of skin to remove orfade a tattoo.

Further areas of applicability will become apparent from the descriptionprovided herein. It should be understood that the description andspecific examples are intended for purposes of illustration only and arenot intended to limit the scope of the present disclosure.

DRAWINGS

The drawings described herein are for illustration purposes only and arenot intended to limit the scope of the present disclosure in any way.

FIG. 1A depicts a photomicrograph representing skin that was treatedwith clodronate liposomes fluorescently labeled with a green dye. InFIG. 1A, the photomicrograph illustrates the dermal layers marked withwhite arrows to indicate tattoo ink taken up by macrophages as seen withlight microscopy.

FIG. 1B depicts a photomicrograph representing skin that was treatedwith clodronate liposomes fluorescently labeled with a green dye. InFIG. 1B, the photomicrograph illustrates the dermal layers marked withwhite arrows to indicate tattoo ink and clodronate liposomes taken up bymacrophages as seen with fluorescence microscopy.

FIG. 1C depicts a photomicrograph representing skin that was treatedwith clodronate liposomes fluorescently labeled with a green dye. InFIG. 1C, the photomicrograph illustrates an overlay of the lightmicroscopy structures with the presence of macrophages havingphagocytosed the clodronate liposomes as seen with fluorescencemicroscopy.

FIG. 1D depicts a photomicrograph representing a lymph node tissuesection in an animal under light microscopy that was treated withclodronate liposomes fluorescently labeled with a green dye 24 hoursprior. In FIG. 1D, the photomicrograph illustrates the lymph node intreated animals 24 hours prior to sacrifice.

FIG. 1E depicts a photomicrograph representing a lymph node tissuesection in an animal that was treated with clodronate liposomesfluorescently labeled with a green dye 24 hours prior. In FIG. 1E, thephotomicrograph illustrates the lymph node viewed under fluorescencemicroscopy to view macrophages indicating digested clodronate liposomesin treated animals 24 hours prior to sacrifice.

FIG. 1F depicts a photomicrograph representing a lymph node tissuesection in an animal that was treated with clodronate liposomesfluorescently labeled with a green dye 2 weeks prior. In FIG. 1F, thephotomicrograph illustrates the lymph node viewed as an overlay underfluorescence microscopy for presence of macrophages having phagocytosedthe clodronate liposomes in treated animals 24 hours prior to sacrifice.

FIG. 1G depicts a photomicrograph representing a lymph node tissuesection in an animal under light microscopy that was treated withclodronate liposomes fluorescently labeled with a green dye 2 weeksprior. In FIG. 1G, the photomicrograph illustrates the lymph node intreated animals 2 weeks to sacrifice.

FIG. 1H depicts a photomicrograph representing lymph node tissue sectionin an animal that was treated with clodronate liposomes fluorescentlylabeled with a green dye 2 weeks prior. In FIG. 1H, the photomicrographillustrates the lymph node viewed under fluorescence microscopy to viewmacrophages indicating digested clodronate liposomes in treated animals2 weeks prior to sacrifice.

FIG. 1I depicts a photomicrograph representing lymph node tissue sectionin an animal that was treated with clodronate liposomes fluorescentlylabeled with a green dye 2 weeks prior. In FIG. 1I, the photomicrographillustrates the lymph node viewed as an overlay under fluorescencemicroscopy for presence of macrophages having phagocytosed theclodronate liposomes in treated animals 2 weeks prior to sacrifice.

FIG. 2 depicts a photomicrograph representing dermal skin tissue in anexperimental animal treated with clodronate liposomes of the presenttechnology and an apoptosis antibody to illustrate the presence ofapoptotic macrophages. As can be seen in FIG. 2, apoptotic macrophagesare seen with digested tattoo ink particles.

FIG. 3 depicts a photomicrograph representing dermal skin tissue in anexperimental animal stained with hematoxylin and eosin that was tattooedand left to heal for 12 weeks.

FIG. 4A depicts a photomicrograph representing a light microscopy imageof a tattooed dermal skin tissue section after 24 hours in anexperimental animal treated with fluorescently labeled clodronateliposomes of the present technology.

FIG. 4B depicts a photomicrograph representing a fluorescence microscopyimage of a tattooed dermal skin tissue section after 24 hours in anexperimental animal treated with fluorescently labeled clodronatecontaining liposomes of the present technology.

FIG. 4C depicts a photomicrograph representing the light microscopyimage overlayed over the fluorescence microscopy image of the tattooeddermal skin tissue section after 24 hours in an experimental animaltreated with fluorescently labeled clodronate containing liposomes ofthe present technology.

FIG. 5A depicts a photomicrograph representing a fluorescence microscopyimage of a tattooed dermal skin tissue section obtained after 24 hoursin an experimental animal treated with fluorescently labeled clodronatecontaining liposomes of the present technology.

FIG. 5B depicts a photomicrograph representing a fluorescence microscopyimage of a tattooed dermal skin tissue section obtained after 2 days inan experimental animal treated with fluorescently labeled clodronatecontaining liposomes of the present technology.

FIG. 5C depicts a photomicrograph representing a fluorescence microscopyimage of a tattooed dermal skin tissue section obtained after 5 days inan experimental animal treated with fluorescently labeled clodronatecontaining liposomes of the present technology.

FIG. 5D depicts a photomicrograph representing a fluorescence microscopyimage of a tattooed dermal skin tissue section obtained after 7 days inan experimental animal treated with fluorescently labeled clodronatecontaining liposomes of the present technology.

FIG. 5E depicts a photomicrograph representing a fluorescence microscopyimage of a tattooed dermal skin tissue section obtained after 14 days inan experimental animal treated with fluorescently labeled clodronatecontaining liposomes of the present technology.

FIG. 6A depicts a photomicrograph representing a light microscopy imageof a tattooed dermal skin tissue section obtained after 2 days posttreatment in an experimental animal treated with clodronate containingliposomes identifying apoptosis in cells of the dermis.

FIG. 6B depicts a photomicrograph representing a light microscopy imageof a lymph node tissue section obtained after 2 days post treatment in atattooed experimental animal treated with clodronate containingliposomes identifying apoptosis in cells of the dermis. Note the lack ofTUNEL staining indicating a lack of apoptotic cells in the lymph node.

FIG. 7 depicts a photomicrograph representing a fluorescence microscopyimage of a lymph node tissue section obtained after 7 days in a tattooedexperimental animal treated with fluorescently labeled clodronateliposomes of the present technology. CD11b+ cells migrating from theskin to the lymph node are identified in the lymph node tissue sectionas shown by positive CD11b staining following treatment with theclodronate containing liposomes of the present technology.

FIG. 8A depicts a photomicrograph representing a light microscopy imageof an untreated tattooed dermal skin tissue section obtained after 7days post tattoo application.

FIG. 8B depicts a photomicrograph representing a light microscopy imageof a tattooed dermal skin tissue section obtained after 7 days posttreatment in an experimental animal treated with clodronate containingliposomes.

FIG. 8C depicts a photomicrograph representing a light microscopy imageof an untreated tattooed dermal skin tissue section obtained after 14days post tattoo application.

FIG. 8D depicts a photomicrograph representing a light microscopy imageof a tattooed dermal skin tissue section obtained after 14 days posttreatment in an experimental animal treated with clodronate containingliposomes.

FIG. 9A depicts a photomicrograph representing a light microscopy imageof a lymph node tissue section obtained after 1 day post treatment in atattooed experimental animal treated with fluorescently labeledclodronate liposomes of the present technology.

FIG. 9B depicts a photomicrograph representing a fluorescence microscopyimage of a lymph node tissue section obtained after 1 day post treatmentin a tattooed experimental animal treated with fluorescently labeledclodronate liposomes of the present technology.

FIG. 9C depicts a photomicrograph representing the light microscopyimage overlayed over the fluorescence microscopy image of a lymph nodetissue section after 1 day post treatment in a tattooed experimentalanimal treated with fluorescently labeled clodronate containingliposomes of the present technology.

FIG. 9D depicts a photomicrograph representing a light microscopy imageof a lymph node tissue section obtained after 14 days post treatment ina tattooed experimental animal treated with fluorescently labeledclodronate liposomes of the present technology.

FIG. 9E depicts a photomicrograph representing a fluorescence microscopyimage of a lymph node tissue section obtained after 14 days posttreatment in a tattooed experimental animal treated with fluorescentlylabeled clodronate liposomes of the present technology.

FIG. 9F depicts a photomicrograph representing the light microscopyimage overlayed over the fluorescence microscopy image of the lymph nodetissue section after 14 days post treatment in a tattooed experimentalanimal treated with fluorescently labeled clodronate containingliposomes of the present technology.

FIG. 9G depicts a photomicrograph representing a fluorescence microscopyimage of a lymph node tissue section obtained after 14 days posttreatment in a tattooed experimental animal treated with fluorescentlylabeled clodronate liposomes of the present technology. Colocalizationof tattoo ink and fluorescently labeled clodronate liposomes/cell debrisare indicated by white arrows.

FIG. 10 depicts a photomicrograph representing a fluorescence microscopyimage of a lymph node tissue section obtained after 14 days posttreatment in a tattooed experimental animal treated with fluorescentlylabeled clodronate liposomes of the present technology and stained withphagocytic cell (monocytes and macrophage) marker CD11b. Colocalizationof fluorescently labeled clodronate liposomes/cell debris and the CD11b+staining are indicated by white arrows.

FIG. 11A depicts a photomicrograph representing a fluorescencemicroscopy image of a lymph node tissue section obtained after 1 daypost treatment in a tattooed experimental animal treated withfluorescently labeled clodronate liposomes of the present technology

FIG. 11B depicts a photomicrograph representing a fluorescencemicroscopy image of a lymph node tissue section obtained after 2 dayspost treatment in a tattooed experimental animal treated withfluorescently labeled clodronate liposomes of the present technology.

FIG. 11C depicts a photomicrograph representing a fluorescencemicroscopy image of a lymph node tissue section obtained after 5 dayspost treatment in a tattooed experimental animal treated withfluorescently labeled clodronate liposomes of the present technology.

FIG. 11D depicts a photomicrograph representing a fluorescencemicroscopy image of a lymph node tissue section obtained after 7 dayspost treatment in a tattooed experimental animal treated withfluorescently labeled clodronate liposomes of the present technology.

FIG. 11E depicts a photomicrograph representing a fluorescencemicroscopy image of a lymph node tissue section obtained after 14 dayspost treatment in a tattooed experimental animal treated withfluorescently labeled clodronate liposomes of the present technology.

FIG. 12A depicts a photomicrograph representing a light microscopy imageof a tattooed dermal skin tissue section harvested after 12 weekstreatment with a liposomal control in an experimental animal. Animalswere treated once per week for 12 weeks

FIG. 12B depicts a photomicrograph representing a light microscopy imageof a tattooed dermal skin tissue section harvested after 12 weekstreatment with clodronate liposomes of the present technology in anexperimental animal. Animals were treated once per week for 12 weeks

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is notintended to limit the present disclosure, application, or uses.

The following description of technology is merely exemplary in nature ofthe subject matter, manufacture and use of one or more inventions, andis not intended to limit the scope, application, or uses of any specificinvention claimed in this application or in such other applications asmay be filed claiming priority to this application, or patents issuingtherefrom. The following definitions and non-limiting guidelines must beconsidered in reviewing the description of the technology set forthherein. All references cited in the “Description” section of thisspecification are hereby incorporated by reference in their entirety.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been made,or tested.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the materials, compositions, devices,and methods of this technology. Similarly, the terms “can” and “may” andtheir variants are intended to be non-limiting, such that recitationthat an embodiment can or may comprise certain elements or features doesnot exclude other embodiments of the present technology that do notcontain those elements or features.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may haveother ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3,3-10, and 3-9.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is use herein to describe and claimthe present invention, the invention, or embodiments thereof, mayalternatively be described using more limiting terms such as “consistingof” or “consisting essentially of” the recited ingredients.

In various embodiments, the present invention provides a method forremoving a tattoo in a region of skin the method comprising:administering to a least a portion of the tattoo a compositioncomprising an effective amount of a bisphosphonate and at least onepharmaceutically acceptable excipient to at least cause fading of thetattoo in the region of skin.

A “tattoo” is a portion of skin in which tattoo ink has been embedded.

In various embodiments, the team “removing a tattoo” from a region ofskin means extracting, for example by displacing, tattoo ink (inkpigment or ink particles) from the region of tissue underlying orsurrounding the tattoo, and includes dislodging and withdrawal of theink particles that is sufficient to cause some fading, with or withouttotal elimination, of the tattoo in the region of skin being treated.“Removal” of a tattoo from a region of skin similarly means havingextracted the tattoo ink from the tissue that is sufficient to causesome fading, with or without total elimination, of the tattoo.

As used herein the term “particle” refers to fully closed carriermolecules that may be phagocytosed by phagocytic cells, including butnot limited to polymeric particles, microcapsules, liposomes,microspheres, microemulsions, nanoparticles, nanocapsules andnanospheres. In various embodiments, bisphosphonate particles areparticles containing one or more bisphosphonate, diphosphonate, oracids, salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof. The bisphosphonate, diphosphonate, or acids, salts,esters, hydrates, polymorphs, hemihydrates, solvates, and derivativesthereof is released from the bisphosphonate particle once thebisphosphonate particle has been engulfed by the phagocytic cell. Invarious embodiments, the bisphosphonate particles of the presenttechnology can measure (diameter and/or length) from about 0.01 micronsto about 30 microns, preferably from about 0.05 microns to about 25microns, or from about 0.1 microns to about 20 microns (μm), or fromabout 0.1 microns to about 10 microns, or from about 0.1 microns toabout 1 micron.

Compositions

The present inventor has identified a class of drugs, bisphosphonates,that are useful in the reduction and destruction of phagocytic cellsthat have taken up tattoo ink pigment and tattoo ink particles therebyreducing the amount of visible ink pigment and particles of a tattoo inthe treated skin. It is believed that during and immediately after thetattoo ink has been embedded into a subject's skin, the phagocytic cellinvolved in the immune response engulf the tattoo ink pigment andparticles and transport it to the draining lymph node via the lymphaticsystem. A lesser proportion of the phagocytic cells remain at the siteof the tattoo in perivascular areas. These phagocytic cells store theindigestible tattoo ink pigment and particles in residual bodies whereit can reside for decades, thus being responsible for the permanency ofa tattoo.

The bisphosphonates as a class of drugs are thought to inhibitosteoclastic bone resorption via a mechanism that differs from that ofother antiresorptive agents. Bisphosphonates are believed attach tohydroxyapatite binding sites on bony surfaces, especially surfacesundergoing active resorption. When osteoclasts begin to resorb bone thatis impregnated with bisphosphonate, the bisphosphonate released duringresorption impairs the ability of the osteoclasts to form the ruffledborder, to adhere to the bony surface, and to produce the protonsnecessary for continued bone resorption. Bisphosphonates also reduceosteoclast activity by decreasing osteoclast progenitor development andrecruitment and by promoting osteoclast apoptosis. In addition to theirinhibitory effect on osteoclasts, bisphosphonates appear to havebeneficial effects on osteoblasts. In a murine model ofglucocorticoid-induced osteoporosis, bisphosphonates prevented osteocyteand osteoblast apoptosis. Bisphosphonates are commonly employedmedically to inhibit bone resorption, and therefore, they find utilityin the treatment of hypercalcemia, osteoporosis, metastatic bonedisease, and Paget disease. Bisphosphonates all have in common the P—C—Pstructure, which is similar to the P—O—P structure of nativepyrophosphate and differ from each other only at the two “R” groups.Some bisphosphonates, for example, neridronate, ibandronate,pamidronate, risedronate, and zoledronic acid have a nitrogen group andare called nitrogen-containing bisphosphonates in contrast to etidronateand tiludronate, which do not.

The terms “bisphosphonate” and “diphosphonate” as used herein, includeacids, salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof.

In several embodiments, bisphosphonate, diphosphonate, or acids, salts,esters, hydrates, polymorphs, hemihydrates, solvates, and derivativesthereof are used in compositions of the present technology to removeresident and non-resident phagocytic cells having digested tattoo inkpigment and particles in the perivascular area of the subject's dermallayers.

Examples of phagocytic cells include, but are not limited to cells ofthe mononuclear phagocytic system, including, but not limited tomacrophages and circulating monocytes. Other cells capable ofphagocytosis include for example neutrophils, dendritic cells, andfibroblasts. Most preferably the phagocytic cells are macrophages and/ormonocytes. According to this aspect of the present invention, inhibitionof phagocytic cells includes reducing the number of, eliminating (i.e.,killing), retarding the proliferation of and/or reducing the activity ofphagocytic cells (e.g. reducing the ability to phagocytose or to secretecytokines). Pharmaceutical agents capable of inhibiting phagocytic cellsare described herein below.

In various embodiments, the compositions of the present technologycontain at least one bisphosphonate, diphosphonate, or acids, salts,esters, hydrates, polymorphs, hemihydrates, solvates, and derivativesthereof selected from: alendronate, cimadronate, incadronate,clodronate, etidronates, risedronate, zoledronate, ibandronate,minodronate, pamidronate, piridronate, tiludronate, olpadronate,neridronate, YH529, EB 1053 and ISA-13-1. At least one of thesebisphosphonate, diphosphonate, or acids, salts, esters, hydrates,polymorphs, hemihydrates, solvates, and derivatives thereof isformulated with at least one pharmaceutically acceptable carrier,excipient or diluent to form a tattoo removal composition that can beapplied to at least a portion of a tattoo by administration methodsdisclosed herein.

In some embodiments, the bisphosphonate, diphosphonate, or acids, salts,esters, hydrates, polymorphs, hemihydrates, solvates, and derivativesthereof can admixed with the at least one pharmaceutically acceptablecarrier, excipient or diluent, or the bisphosphonate, diphosphonate, oracids, salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof can be encapsulated in a particle, thereby forming abisphosphonate particle. As used herein, a bisphosphonate particle caninclude: liposomes, nanospheres, microspheres, lipid bilayers,nanoparticles, microparticles, microcapsules, nanocapsules, and thelike. The particle may have an outer wall defining an interior spacethat can be filled with the bisphosphonate, diphosphonate, or acids,salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof. In some embodiments, the particle may comprise atleast one biodegradable material, for example, one or more lipids (forexample a liposome forming lipid), PGLA, silicon, cellulose, inorganicminerals (for example, calcium based ceramics) that degrade whentaken-up or phagocytosed by a phagocytic cell. As used herein, the termphagocytosis also encompasses forms of endocytosis, including but notlimited to pinocytosis, receptor-mediated endocytosis and other cellularmeans for absorbing/internalizing tattoo ink pigment or ink particlesmaterial in the dermal layers of the skin.

Dosage amount of the bisphosphonate, diphosphonate, or acids, salts,esters, hydrates, polymorphs, hemihydrates, solvates, and derivativesthereof and interval may be adjusted individually to provide skinintradermal levels of the bisphosphonate, diphosphonate, or acids,salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof that are sufficient to induce the biological effect(effective concentration, EC). The EC will vary for each preparation,but can be estimated from in vitro data. Dosages necessary to achievethe EC will depend various factors related to the subject being treatedand route of administration. Detection assays can be used to determineplasma concentrations of the liposomes and/or levels of the administeredbisphosphonate, diphosphonate, or acids, salts, esters, hydrates,polymorphs, hemihydrates, solvates, and derivatives thereof.

In some embodiments, the compositions of the present technology may beadministered to a region of skin using an injectable means. In someembodiments, an injectable composition can comprise bisphosphonateparticles containing at least one bisphosphonate, diphosphonate, oracids, salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof and at least one pharmaceutically acceptableexcipient. In various embodiments, bisphosphonate particles can includeliposomes, nanospheres, microspheres, lipid bilayers, nanoparticles,microparticles, microcapsules, nanocapsules, and the like.

According to this aspect of the present technology, bisphosphonateparticles are prepared so that the size of the bisphosphonate particleis large enough to essentially be internalized by phagocytosis, thusdirecting the compositions of the present technology specifically tophagocytic cells. Bisphosphonate particles ranging from about 0.01 μm toabout 5.0 μm, preferably from about 0.05 μm to about 1 μm can beemployed in the present methods. In some embodiments, bisphosphonate,diphosphonate, or acids, salts, esters, hydrates, polymorphs,hemihydrates, solvates, and derivatives thereof containing particles aretargeted directly to phagocytic cells in the dermal layers of thesubject's skin are preferably in the size range of 0.02-2.5 μm, morepreferably 0.05-1.0 μm and more preferably 0.07-0.7 μm.

The bisphosphonate particle may be provided in any suitable formincluding but not limited to a solution, a suspension, an emulsion, orany form of mixture. The bisphosphonate particle may be delivered informulation with any pharmaceutically acceptable excipient, carrier, orvehicle. For example, the formulation may be delivered in a conventionaltopical dosage form such as, for example, a cream, an ointment, anaerosol formulation, a non-aerosol spray, a gel, a foam, a solution, asuspension, a dispersion, an emulsion, a microemulsion, a paste, apowder, a solid stick (e.g., wax- or petroleum-based sticks), a wipe, anoil, a lotion, and the like. In one particular embodiment, thebisphosphonate particle is provided in a cream formulation suitable fortopical administration. In another embodiment, the bisphosphonateparticle is provided as an injectable formulation, for example, anintradermal formulation.

In various embodiments, the bisphosphonate particle can comprise one ormore bisphosphonates in admixture with a pharmaceutically acceptableexcipient. Pharmaceutically acceptable excipients for use withbisphosphonate particles may include one or more of a carrier, adiluent, a binder, a lipid, for example, cholesterol, olive oil etc., aliposome forming lipid, a drug eluting polymer or monomer, a skinpenetration enhancer, for example a lower alcohol (e.g. methanol,ethanol, n-propanol, isopropanol, or combinations thereof), a chelatingagent, a surfactant, an emulsifying agent, a thickening agent, a pHmodifier, a pain reliever, an antibiotic, an anti-inflammatory agent, ananesthetic, a steroid, and a chelating agent (for example: ethylenediaminetetraacetate (EDTA), desferrioxamine, clioquinol, ethylene glycoltetraacetic acid (EGTA), small hydrophobic chelators such asphenanthroline or bipyridine, hexadentate iron chelator and deferoxamine(also known as desferrioxamine, desferoxamine, DFO, DFOA or desferal).

In a specific embodiment, the bisphosphonate particle is a liposomecontaining one or more bisphosphonate, diphosphonate, or acids, salts,esters, hydrates, polymorphs, hemihydrates, solvates, or derivativesthereof. In this embodiment, liposomes useful in the present technologycan include any synthetic structure (unilamellar, or multilamellarvesicles) that is made with liposomal lipids in a liquid crystallinephase or a liquid gel phase, which enclose a volume of liquid comprisinga bisphosphonate, diphosphonate, or acids, salts, esters, hydrates,polymorphs, hemihydrates, solvates, and derivatives thereof. In someembodiments, the liposomes may be coated (for example, with albumin) oruncoated.

In some embodiments, liposomes can include emulsions, foams, micelles,insoluble monolayers, liquid crystals, phospholipid dispersions,lamellar layers and the like. The liposomes may be prepared by any ofthe known methods in the art. The liposomes may be positively charged,neutral or, more preferably, negatively charged. In various embodiments,the liposome can comprise cholesterol, a liposome forming lipid andoptionally, a polyionic polymer. In some of these embodiments, theliposome forming lipid selected from the group consisting ofhydrogenated soy phosphatidylcholine, distearoylphosphatidylcholine,sphingomyelin, diacylglycerol, phosphatidyl ethanolamine,phosphatidylglycerol, phosphatidylserine, distearylphosphatidylcholineand distearylphosphatidyl ethanolamine and combinations thereof,

In various embodiments, the liposomes of the present technology may alsocomprise a non-ionic surfactant. In some examples the non-ionicsurfactant can include: polyoxyl 35, polyoxyl 40 hydrogenated castor oil(Cremophor RH 40), and polyoxyl 60 hydrogenated castor oil (Cremophor RH60), as well as d-α-tocopherol, polyethylene glycol 1000 succinate,polysorbate 20, polysorbate 80, Sorbitan-monolaurate (Span 20), Sorbitanmonopalmitate (Span 40); Sorbitan monostearate (Span 60);Sorbitan-monooleate (Span 80), Solutol HS 15, poloxamer 407, LabrafilM-1944CS, Labrafil M-2125CS, Labrasol, Gellucire 44/1. In someembodiments, the non-ionic surfactant is sorbitan monooleate.

As detailed above, many properties influence uptake of liposomes byphagocytic cells including, but not limited to liposome size, charge andhydrophobicity, as well as the phospholipids and non-phospholipidcomponents of the liposome.

The liposomes may be modified in any other way to enhance their uptakeby the phagocytic cells, e.g. by attaching to them molecules recognizedselectively by phagocytic cells such as ligands that interact with themacrophage Fe receptor, serum proteins such as albumin, or galactosylligands, or inclusion of substances in the bilayer such as complement,fibronectin, lipoproteins, albumins or gamma globulin.

The liposomes may be a single lipid layer or may be multilamellar. Ifthe agent capable of inhibiting phagocytic cells is hydrophilic, itsdelivery may be further improved using large unilamellar vesiclesbecause of their greater internal volume. Conversely, if the agent ishydrophobic, its delivery may be further improved using multilamellarvesicles. Alternatively, the agent capable of down-regulating phagocyticcells (e.g. oligonucleotide) may not be able to penetrate the lipidbilayer and consequently would remain adsorbed to the liposome surface.In this case, increasing the surface area of the liposome may furtherimprove delivery of the therapeutic agent. Suitable liposomes inaccordance with the invention are preferably non-toxic liposomes suchas, for example, those prepared from any one or more ofphosphatidylcholine, distearoylphosphatidylcholine, sphingomyelin,diacylglycerol, phosphatidyl ethanolamine, phosphatidylglycerol,distearylphosphatidylcholine and distearylphosphatidyl ethanolamine, andcholesterol. The diameter of the liposomes used preferably ranges fromabout 0.05-25 microns, for example, from about 0.1 microns to about 10microns, or from about 0.08 to about 1.0 micron. However, other liposomesize or diameter ranges suitable for phagocytosis by phagocytic cellsmay also be used, and can vary greatly between cell types, such asmonocytes and macrophages. In some illustrative embodiments, theliposomes can range in diameters from between, for example, 0.01 micronto about 20 microns, or for example, from about 0.1 micron to about 10microns, or from about 0.008 microns to about 1 micron. For sizingliposomes, homogenization may be used, which relies on shearing energyto fragment large liposomes into smaller ones. Homogenizers, which maybe conveniently used, include microfluidizers produced by Microfluidicsof Boston, Mass. In a typical homogenization procedure, liposomes arerecirculated through a standard emulsion homogenizer until selectedliposomes sizes are observed. The particle size distribution can bemonitored by conventional laser beam particle size discrimination.Extrusion of liposomes through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is an effective method for reducing liposomesizes to a relatively well defined size distribution. Typically, thesuspension is cycled through the membrane one or more times until thedesired liposome size distribution is achieved. The liposomes may beextruded through successively smaller pore membranes to achieve agradual reduction in liposome size.

In some embodiments, the liposomes of the present technology comprise abisphosphonate, for example, clodronate, encapsulated in a liposome madefrom hydrogenated soy phosphatidylcholine as the liposome forming lipid.The clodronate liposomes may also contain sorbitan monooleate. Methodsfor making clodronate containing liposomes are known, for example, asprovided in Van Rooijen, N, Sanders, A. (1994) Liposome mediateddepletion of macrophages: mechanism of action, preparation of liposomesand applications J. Immunol. Methods 174:83-93, the disclosure of whichis hereby incorporated by reference in its entirety. In someembodiments, Clodronate-liposomes can be produced by admixture of aliposome forming lipid with cholesterol and a solution of clodronate(ranging from about 0.1 to about 1.5 M) and sonicating the admixturegently. The resultant liposomes can then be dialyzed, centrifuged, orotherwise, washed to remove free clodronate prior to administration orformulation into a topical, transdermal or injectable dose. In someembodiments, injection ready clodronate liposomes (multilamellarclodronate encapsulated liposomes) are commercially available fromEncapsula NanoSciences, (Brentwood, Tenn. USA).

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc. The concentration of bisphosphonate particles in these formulationscan vary widely, i.e., from less than about 0.5%, usually at or at leastabout it to as much as 15 or 20% by weight and will be selectedprimarily by fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected.

In one embodiment, the entrapped, encapsulated or capturedbisphosphonate used in connection with the formulations and methods ofthe present technology may have a concentration ranging from about 0.7Mto about 0.001M. In some embodiments, the amount of the bisphosphonaterelative to the amount of the particle may represent from about fromless than about 0.5%, to about 20% of the particle (e.g. liposome) on amolar basis and 0.05 to 10% on a per weight basis. Described by ratio,in some embodiments, liposome particles have an encapsulatedbisphosphonate to lipid ratio (on a molar basis) of about 1:3 to 1:250and an encapsulated bisphosphonate to lipid ratio (on a per weightbasis) of about 1:10 to 1:2000. In some embodiments, the molar ratio ofencapsulated bisphosphonate, for example, clodronate, to lipid in theliposomal preparation can range from 1:4 to 3:4. In some embodiments,the weight ratio of bisphosphonate to lipid can range from 1:3 to 1:10.In various embodiments, the bisphosphonate particles can be formulatedto produce a cream, an ointment, an aerosol formulation, a non-aerosolspray, a gel, a foam, a solution, a suspension, a dispersion, anemulsion, a microemulsion, a paste, a powder, a solid stick (e.g., wax-or petroleum-based sticks), a wipe, an oil, a lotion, such that whenadministered to a region of skin, wither through a topical application,a transdermal device, such as a patch or injected intradermally, theadministered dose of the bisphosphonate particle formulation providesfrom about 1 nanogram of bisphosphonate per 50 micrograms of intradermaltissue to about 25 micrograms of bisphosphonate per 50 micrograms oftissue on a per weight basis. In other embodiments, the formulations ofthe present technology comprise from about 2% to about 20% (% wt/vol.)of the liposomes, wherein the ratio of bisphosphonate to lipid in theliposomes ranges from 1:3 to 1:250 on a per weight basis.

In some embodiments, the various liposomal compositions compriseliposomes containing 0.1 mg/mL to about 1,000 mg/mL of thebisphosphonate, for example, clodronate within the liposomes,preferably, from about 1 mg/mL to about 500 mg/mL. In some embodiments,the molar ratio of bisphosphonate, for example, clodronate, to lipid inthe liposome can range from about 1:4 to about 3:4. In variousembodiments, the maximum solubility of the bisphosphonate used, forexample, clodronate, can range from about 100 mg/mL to about 300 mg/mL.In various embodiments, the liposome particles of the present inventionentrap about 0.5% to about 5%, or from about 1% to about 2% of abisphosphonate, for example, a clodronate solution (having aconcentration of about 200 mg/mL to about 300 mg/mL). In variousembodiments, the weight ratio of bisphosphonate to lipid in the preparedliposomes can range from about 1:3 to about 1:10. In some embodiments,the molar ratio of encapsulated bisphosphonate, for example, clodronate,to lipid in the liposomal preparation can range from about 1:4 to about3:4. In some embodiments, the weight ratio of bisphosphonate, forexample, clodronate to lipid can range from 1:2 to 1:10, for example,from about 1:3 to about 1:10.

Any method known in the art can be used to determine the size of theparticle before administration to a patient in need thereof. Forexample, a Nicomp Submicron Particle Sizer (model 370, Nicomp, SantaBarbara, Calif.) utilizing laser light scattering can be used. Othermethods of sizing particles are also known in the art.

Determination of the optimal size, formulation and/or amount, of aparticle to be engulfed by a phagocytic cell may be determined usingprocedures known in the art such as the assays described in U.S. Pat.Appl. No. 20040266734 and U.S. Pat. Appl. No. 20040266734; and inDanenberg et al., Journal of cardiovascular pharmacology 2003, 42:671-9;Circulation 2002, 106:599-605; Circulation 2003, 108:2798-804. In an invitro screening assay, liposome uptake can be visually inspected usingin-vitro tissue culture of macrophages. The phagocytic cells may beobtained from an established cell line or isolated from an individual asa primary cell line. In an in vivo assay, bisphosphonate particles canbe administered to a test subject (e.g. an experimental subject, amouse, or a rabbit) and after a set amount of time, tissues may beremoved and examined using confocal microscopy for evidence ofphagocytic cell ingestion of the bisphosphonate particles and phagocyticcell depletion. Similarly, depletion of phagocytic cells containing inkpigment or particles may similarly be visualized using confocalmicroscopy.

Typically, particles of the present invention sequester thebisphosphonate, diphosphonate, or acids, salts, esters, hydrates,polymorphs, hemihydrates, solvates, and derivatives thereof, capable ofinhibiting phagocytic cells for a sufficient time to enhance delivery ofthe bisphosphonate, diphosphonate, or acids, salts, esters, hydrates,polymorphs, hemihydrates, solvates, and derivatives thereof, and becomebioavailable when they are digested by phagocytic cells in theperivascular area of the subject's treated skin. Furthermore, thebisphosphonate, diphosphonate, or acids, salts, esters, hydrates,polymorphs, hemihydrates, solvates, and derivatives thereof is typicallyreleased from the bisphosphonate particles when they are ingested withinthe target cell (e.g., the phagocytic cell) at the target site.

In addition to solutions, suspensions, dispersions, emulsion,microemulsions, pastes, powders that can be injected intradermally, thebisphosphonate particles can also be formulated and administered astopical formulations such as creams, ointments, aerosol formulations, anon-aerosol sprays, gels, foams, and any other known and medicallyacceptable topical formulations, which are known in the art anddescribed in “Remington: The Science And Practice Of Pharmacy”,1577-1591, 1672-1673, 866-885 (Alfonso R. Gennaro ed. 19th ed. 1995);Ghosh, T. K.; et al. Transdermal And Topical Drug Delivery Systems(1997), both of which are hereby incorporated herein by reference.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Specific formulationis dependent upon the route of administration chosen, and the agent. Inone embodiment, the composition is applied topically to a region of skincontaining at least a portion of a tattoo. A topically administrablecomposition that can be used in embodiments of the present inventioncomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a bisphosphonate, diphosphonate, or acids, salts,esters, hydrates, polymorphs, hemihydrates, solvates, and derivativesthereof. The carriers useful for topical delivery of the specifiedcompounds according to embodiments of the invention can be any carrierknown in the art for topically administering pharmaceuticals, including,but not limited to, pharmaceutically acceptable solvents, such as apolyalcohol or water; emulsions (either oil-in-water or water-in-oilemulsions), such as creams or lotions; micro emulsions; gels; ointments;liposomes; nanoparticles or microparticles, powders; and aqueoussolutions or suspensions. The pharmaceutically acceptable carrier one ormore inactive pharmaceutically acceptable excipients, including, but notlimited to, binders, carriers, diluents, suspending agents, lubricants,emulsifiers, flavorants, preservatives, dyes, and coatings.

The topically administrable composition are prepared by mixing apharmaceutically acceptable carrier with a therapeutically effectiveamount of a bisphosphonate, diphosphonate, or acids, salts, esters,hydrates, polymorphs, hemihydrates, solvates, and derivatives thereof,according to known methods in the art, for example, methods provided bystandard reference texts such as, Remington: The Science And Practice OfPharmacy 1577-1591, 1672-1673, 866-885 (Alfonso R. Gennaro ed. 19th ed.1995); Ghosh, T. K.; et al. Transdermal And Topical Drug DeliverySystems (1997), both of which are hereby incorporated herein byreference.

In one embodiment, the topically administrable composition is in theform of an emulsion. Emulsions, such as creams and lotions are suitabletopical formulations for use in the invention. An emulsion is adispersed system comprising at least two immiscible phases, one phasedispersed in the other as droplets ranging in diameter from 0.1 μm to100 μm. An emulsifying agent is typically included to improve stability.When water is the dispersed phase and oil is the dispersion medium, theemulsion is termed a water-in-oil emulsion. When oil is dispersed asdroplets throughout the aqueous phase as droplets, the emulsion istermed an oil-in-water emulsion. Emulsions, such as creams and lotionsthat can be used as topical carriers and their preparation are disclosedin Remington: The Science and Practice of Pharmacy 282-291 (Alfonso R.Gennaro ed. 19th ed. 1995), hereby incorporated herein by reference.

In another embodiment, the topically administrable composition is in theform of a gel, for example, a two-phase gel or a single-phase gel. Gelsare semisolid systems consisting of suspensions of small inorganicparticles or large organic molecules interpenetrated by a liquid. Whenthe gel mass comprises a network of small discrete inorganic particles,it is classified as a two-phase gel. Single-phase gels consist oforganic macromolecules distributed uniformly throughout a liquid suchthat no apparent boundaries exist between the dispersed macromoleculesand the liquid. Suitable gels for use in the invention are disclosed inRemington: The Science and Practice of Pharmacy 1517-1518 (Alfonso R.Gennaro ed. 19th ed. 1995), hereby incorporated herein by reference.

In an embodiment, the topically administrable composition comprises anaqueous gel comprising water and a water-gelling amount of apharmaceutically acceptable gelling agent selected from the groupconsisting of carbomers, glycerin polyacrylate, and mixtures thereof,and the topical composition has a physiologically acceptable pH.

Polymer thickeners (gelling agents) that may be used in compositionsaccording to embodiments of the present invention include those known toone skilled in the art, such as hydrophilic and hydroalcoholic gellingagents frequently used in the cosmetic and pharmaceutical industries.Preferably, the hydrophilic or hydroalcoholic gelling agent comprises“CARBOPOL®” (B.F. Goodrich, Cleveland, Ohio), “HYPAN®” (KingstonTechnologies, Dayton, N.J.), “NATROSOL®” (Aqualon, Wilmington, Del.),“KLUCEL®” (Aqualon, Wilmington, Del.), or “STABILEZE®” (ISPTechnologies, Wayne, N.J.). Preferably the gelling agent comprisesbetween about 0.2% to about 4% by weight of the composition. Moreparticularly, the preferred compositional weight percent range for“CARBOPOL®” is between about 0.5% to about 2%, while the preferredweight percent range for “NATROLSOL®” and “KLUCEL®” is between about0.5% to about 4%. The preferred compositional weight percent range forboth “HYPAN®” and “STABILEZE®” is between 0.5% to about 4%.

“CARBOPOL®” is one of numerous cross-linked acrylic acid polymers thatare given the general adopted name carbomer. These polymers dissolve inwater and form a clear or slightly hazy gel upon neutralization with acaustic material such as sodium hydroxide, potassium hydroxide,triethanolamine, or other amine bases. “KLUCEL®” is a cellulose polymerthat is dispersed in water and forms a uniform gel upon completehydration. Other preferred gelling polymers includehydroxyethylcellulose, cellulose gum, MVE/MA decadiene crosspolymer,PVM/MA copolymer, or a combination thereof.

In another preferred embodiment, the topically administrable compositionis in the form of an ointment. Ointments are oleaginous semisolids thatcontain little if any water. Preferably, the ointment is hydrocarbonbased, such as a wax, petrolatum, or gelled mineral oil. Suitableointments for use in the invention are well known in the art and aredisclosed in Remington: The Science and Practice of Pharmacy 1585-1591(Alfonso R. Gennaro ed. 19th ed. 1995), hereby incorporated herein byreference.

In an embodiment of the present invention, the topically administrablecomposition comprises at least one of a cream, an emulsion, a foam, anointment, a dispersion, a paste, a spray, a solution, an oil, or amicroemulsion, wherein the topical compositions comprises abisphosphonate particle and at least one agent selected from the groupconsisting of: stearic acid, sorbitan monooleate (Span80), stearylalcohol, cetyl alcohol, ethanol, glycerin, polyethylene glycol, water,and mixtures thereof, and the topical composition has a physiologicallyacceptable pH.

In another embodiment, the topically administrable composition is in theform of an aqueous solution or suspension, preferably, an aqueoussolution. Suitable aqueous topical formulations for use in the inventioninclude those disclosed in (Alfonso R. Gennaro ed. 19th ed. 1995),hereby incorporated herein by reference.

The pH of the topical formulations of the invention is preferably withina physiologically acceptable pH, e.g., within the range of about 6 toabout 8, more preferably, of about 6.3 to about 6.5. To stabilize thepH, preferably, an effective amount of a buffer is included. In oneembodiment, the buffering agent is present in the aqueous topicalformulation in an amount of from about 0.05 to about 1 weight percent ofthe formulation. Acids or bases can be used to adjust the pH as needed.

Tonicity-adjusting agents can be included in the aqueous topicalformulations to be used in embodiments of the present invention.Examples of suitable tonicity-adjusting agents include, but are notlimited to, sodium chloride, potassium chloride, mannitol, dextrose,glycerin, polyethylene glycol, and propylene glycol. The amount of thetonicity agent can vary widely depending on the formulation's desiredproperties. In one embodiment, the tonicity-adjusting agent is presentin the aqueous topical formulation in an amount of from about 0.5 toabout 0.9 weight percent of the formulation.

Preferably, the aqueous topical formulations have a viscosity in therange of from about 15 cps to about 25 cps. The viscosity of aqueoussolutions of the invention can be adjusted by adding viscosity adjustingagents, for example, but not limited to, polyvinyl alcohol, povidone,hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, orhydroxyethyl cellulose.

In a preferred embodiment, the aqueous topical formulation is isotonicsaline comprising a preservative, such as benzalkonium chloride orchlorine dioxide, a viscosity-adjusting agent, such as polyvinylalcohol, and a buffer system such as sodium citrate and citric acid.

The topically administrable composition can comprise pharmaceuticallyacceptable excipients such as those listed in Remington: The Science AndPractice Of Pharmacy 866-885 (Alfonso R. Gennaro ed. 19th ed. 1995;Ghosh, T. K.; et al. Transdermal And Topical Drug Delivery Systems(1997), hereby incorporated herein by reference, including, but notlimited to, protectives, adsorbents, demulcents, emollients,preservatives, antioxidants, moisturizers, buffering agents,solubilizing agents, skin-penetration agents, and surfactants.

Suitable protectives and adsorbents include, but are not limited to,dusting powders, zinc stearate, collodon, dimethicone, silicones, zinccarbonate, aloe vera gel and other aloe products, vitamin E oil,allatoin, glycerin, petrolatum, and zinc oxide.

Suitable demulcents include, but are not limited to, benzoin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, and polyvinylalcohol.

Suitable emollients include, but are not limited to, animal andvegetable fats and oils, myristyl alcohol, alum, and aluminum acetate.

In an embodiment of the present invention, the topically administrablecomposition further comprises one or more agent selected from the groupconsisting of a preservative, a local anesthetic and a skin humectant.

Suitable preservatives include, but are not limited to, quaternaryammonium compounds, such as benzalkonium chloride, benzethoniumchloride, cetrimide, dequalinium chloride, and cetylpyridinium chloride;mercurial agents, such as phenylmercuric nitrate, phenylmercuricacetate, and thimerosal; alcoholic agents, for example, chlorobutanol,phenylethyl alcohol, and benzyl alcohol; stabilized chlorine dioxide,for example, the proprietary stabilized chlorine dioxide of BioCideInternational, Inc. of Norman, Okla., sold under the trademark Purogene™or Purite™. Other suitable stabilized chlorine dioxide products includethat sold under the trademark DuraKlor® by Rio Linda Chemical Company,Inc., and that sold under the trademark Antheium Dioxide® byInternational Dioxide, Inc. antibacterial esters, for example, esters ofparahydroxybenzoic acid; and other anti-microbial agents such aschlorhexidine, chlorocresol, benzoic acid and polymyxin.

Suitable antioxidants include, but are not limited to, ascorbic acid andits esters, sodium bisulfite, butylated hydroxytoluene, butylatedhydroxyanisole, tocopherols, and chelating agents like EDTA and citricacid.

Suitable moisturizers include, but are not limited to, glycerin,sorbitol, polyethylene glycols, urea, and propylene glycol.

Suitable buffering agents for use with the invention include, but arenot limited to, acetate buffers, citrate buffers, phosphate buffers,lactic acid buffers, and borate buffers.

Suitable solubilizing agents include, but are not limited to, quaternaryammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, sorbitanesters, and polysorbates.

Suitable skin-penetration agents include, but are not limited to, ethylalcohol, isopropyl alcohol, octylphenylpolyethylene glycol, oleic acid,polyethylene glycol 400, propylene glycol, N-decylmethylsulfoxide, fattyacid esters (e.g., isopropyl myristate, methyl laurate, glycerolmonooleate, and propylene glycol monooleate); and N-methylpyrrolidone.

The topically administrable composition according to embodiments of thepresent technology can include pharmaceuticals or their pharmaceuticallyacceptable salts, such as a bisphosphonate, diphosphonate, or acids,salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof, and optionally one or more other pharmaceuticallyactive ingredients, including, but not limited to, coal tar, dithranol(anthralin), corticosteroids like desoximetasone (Topicort),fluocinonide, vitamin D analogs (for example, calcipotriol), retinoids,Argan oil, psoralen, methotrexate, cyclosporine, retinoids or othersynthetic forms of vitamin A, which may aid in the removal of phagocyticcells in the area of administration.

The topically administrable composition according to embodiments of theinvention can further include local anesthetics and analgesics, such ascamphor, menthol, lidocaine, and dibucaine, and pramoxine; antifungals,such as ciclopirox, chloroxylenol, triacetin, sulconazole, nystatin,undecylenic acid, tolnaftate, miconizole, clotrimazole, oxiconazole,griseofulvin, econazole, ketoconozole, and amphotericin B; antibioticsand anti-infectives, such as mupirocin, erythromycin, clindamycin,gentamicin, polymyxin, bacitracin, and silver sulfadiazine; andantiseptics, such as iodine, povidine-iodine, benzalkonium chloride,benzoic acid, chlorhexidine, nitrofurazine, benzoyl peroxide, hydrogenperoxide, hexachlorophene, phenol, resorcinol, and cetylpyridiniumchloride.

In various embodiments, the amount of active agent in the compositionsdescribed herein (i.e. in injectable formulations, topical formulationsand transdermal formulations), can range from 0.01% to 5% by weight ofthe bisphosphonate, diphosphonate, or acids, salts, esters, hydrates,polymorphs, hemihydrates, solvates, and derivatives thereof. Forexample, the composition can comprise, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% 5%, 6%, 7%, 8% 9%,10%, 12%, 14% 16%, 18%, or 20% by weight or by volume of the finalcomposition, of the bisphosphonate, diphosphonate, or acids, salts,esters, hydrates, polymorphs, hemihydrates, solvates, and derivativesthereof.

In a preferred embodiment, the composition comprises 0.05%-20%, 0.1%40%or 0.1-5% by weight of the bisphosphonate, diphosphonate, or acids,salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof.

In several embodiments, the compositions of the present invention cancomprise from about 0.1 mg/mL to about 100 mg/mL of the bisphosphonate,diphosphonate, or acids, salts, esters, hydrates, polymorphs,hemihydrates, solvates, and derivatives thereof, and comprises at leastone liposome forming lipid selected from the group consisting ofhydrogenated soy phosphatidylcholine, distearoylphosphatidylcholine,sphingomyelin, diacylglycerol, phosphatidyl ethanolamine,phosphatidylglycerol, distearylphosphatidylcholine anddistearylphosphatidyl ethanolamine and combinations thereof, and anemulsifier.

Methods of Use

In some embodiments, the present methods involve the administration ofan effective amount of a bisphosphonate or diphosphonate, as usedherein, which include acids, salts, esters, hydrates, polymorphs,hemihydrates, solvates, and derivatives thereof to a region of skincontaining at least a portion of a tattoo to be removed or faded.

The methods of the present invention may be performed on any suitablesubject. Suitable subjects include, but are not limited to, mammals,such as, but not limited to, humans, non-human primates, laboratoryanimals such as mice, rats, guinea-pigs, rabbits, hamsters, ferrets andthe like, companion animals, such as dogs, cats, and stock animals suchas cows, pigs, horses, sheep, goats and the like. In some embodiments,the subject is a human subject.

In some embodiments, the method of the present technology providesremoval or fading of a tattoo in a region of skin, the methodcomprising: administering to a least a portion of the tattoo acomposition comprising an effective amount of a bisphosphonate and atleast one pharmaceutically acceptable excipient to at least cause fadingof the tattoo in said region.

In various embodiments, the administered effective amount of abisphosphonate and at least one pharmaceutically acceptable excipient,is done with the use of a pharmaceutically acceptable composition thatprovides a suitable benefit to risk ratio that is commensurate withstandard medical practices for the dermatological removal of tattoos.

As stated above, useful compositions of the present methods can utilizebisphosphonate, and/or particles containing at least one bisphosphonate,or a diphosphonate, or pharmaceutically acceptable acids, salts, esters,hydrates, polymorphs, hemihydrates, solvates, and derivatives thereof.In some embodiments, bisphosphonates, when encapsulated in liposomes ormicroparticles, or nanoparticles in a “particle” dosage form, they aretaken-up, by the phagocytic process mediated by the macrophages andmonocytes, and to some extent by other cells with phagocytic activitysuch as fibroblasts residing in the dermal layers of the subject's skin.In one embodiment, administration of bisphosphonate particles in theform of liposomes, once inside the phagocytic cells, the liposomalstructure of the liposome is disrupted and the encapsulatedbisphosphonates, diphosphonate, or pharmaceutically acceptable acids,salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof are released into the cytosol of the phagocyticcell, thereby killing the phagocytic cells in the perivascular area ofthe tattoo site. Since macrophages, in their normal state, are recruitedto the dermal layers, they participate in phagocytosis to remove tattooink pigments and particles.

Without wishing to be bound to any particular theory, it is believedthat the administration of the bisphosphonate compositions containingone or more bisphosphonates or diphosphonate, acids, salts, esters,hydrates, polymorphs, hemihydrates, solvates, and derivatives thereof,or particles containing one or more bisphosphonates or diphosphonate,acids, salts, esters, hydrates, polymorphs, hemihydrates, solvates, andderivatives thereof, reduce or fade the visible tattoo by depletingand/or destroying phagocytic cells that have phagocytosed tattoo ink,and/or are important in the accumulation of tattoo ink pigments andparticles as foreign matter. Without wishing to be limited to any oneparticular theory, it is believed that destruction of these residualphagocytic cells containing tattoo ink pigment and/or ink particles inthe perivascular areas of the dermis results in the tattoo ink pigmentand ink particles being transported to the lymphatic nodes for furtherdisposal resulting in the fading of the tattoo in the area treated withthe compositions of the present technology.

Other cells capable of phagocytosis include for example neutrophils,dendritic cells, and fibroblasts. Most preferably the phagocytic cellsare macrophages and/or monocytes. In some embodiments, the use of thecompositions described herein containing one or more bisphosphonates ordiphosphonates, acids, salts, esters, hydrates, polymorphs,hemihydrates, solvates, and derivatives thereof to remove macrophagesand/or monocytes from the dermal layers can occur when the tattoo isfreshly applied to a region of skin (i.e. within 24-72 hours) or afterthe tattoo has matured (from 2 weeks to 50 years).

Liposomes have the benefit of extending half-life by protectingclodronate from decomposition, while also enhancing the specificity formacrophages. Macrophages readily engulf and ingest clodronate liposomes,subsequently releasing the clodronate into the cytosol. Clodronate isincorrectly recognized by the phagocytic cell in the cell'smitochondria, ultimately leading to cell death. In turn, the pigmentparticles contained within the macrophages is released and fragmentedand is disposed in the subject's lymph nodes.

Administration

In accordance with the present technology, a bisphosphonate,diphosphonate, or pharmaceutically acceptable acids, salts, esters,hydrates, polymorphs, hemihydrates, solvates, and derivatives thereof isadministered to a region of skin containing at least a portion of atattoo for removing or fading a tattoo in the region of skin.

The present invention thus provides the use of bisphosphonate, ordiphosphonate, a complex of bisphosphonate, or diphosphonate, or apharmaceutically acceptable acid, salt, ester, hydrate, polymorph,hemihydrate, solvate, and derivative thereof or particles containingsaid bisphosphonate, or diphosphonate, a complex of bisphosphonate, ordiphosphonate, or a pharmaceutically acceptable acid, salt, ester,hydrate, polymorph, hemihydrate, solvate, and derivative thereof; forthe preparation of a composition for removing or fading a tattoo in aregion of skin. In one embodiment, the composition comprises a“particle” dosage form, wherein the bisphosphonate, or diphosphonate, ora pharmaceutically acceptable acid, salt, ester, hydrate, polymorph,hemihydrate, solvate, and derivative thereof is encapsulated, embedded,and/or adsorbed within a particle, dispersed in the particle matrix,adsorbed or linked on the particle surface, or in combination of any ofthese forms. The particle includes any one of liposomes, microparticles,nanoparticles, nanospheres, microspheres, microcapsules, or nanocapsulesknown in the art and examples described herein, or combinations thereof.

In some embodiments, the bisphosphonate containing particles may beadministered in the form of an injectable intradermal solution,emulsion, suspension, dispersion and the like, or administered as atopical composition or as part of a transdermal applicator, such as apatch, to a region of skin containing at least a portion of a tattoo. Insome embodiments, the compositions are formulated for administrationusing an injectable solution containing the inventive bisphosphonateparticles. In some embodiments, the composition is injected into aregion of skin comprising at least a portion of a tattoo intradermallyor by intradermal delivery.

In various embodiments of the present technology, the bisphosphonateparticles are delivered into the intradermal compartment or areintradermally delivered in a region of skin comprising at least aportion of a tattoo. In this manner, the bisphosphonate particles areadministered into the dermis in such a manner that the bisphosphonateparticles readily reach the richly vascularized papillary dermis and aretaken up by the phagocytic cells residing in the perivascular areas ofthe papillary dermis. In some embodiments the bisphosphonatecompositions of the present technology can be placed in the upper regionof the dermis, i.e., the papillary dermis or in the upper portion of therelatively less vascular reticular dermis such that the agent readilydiffuses into the papillary dermis.

In some embodiments, the bisphosphonate particles of the presenttechnology can be delivered predominately at a depth of at least about0.3 mm, more preferably, at least about 0.4 mm and most preferably atleast about 0.5 mm up to a depth of no more than about 2.5 mm, morepreferably, no more than about 2.0 mm and most preferably no more thanabout 1.7 mm. In some embodiments, liposomal bisphosphonate particlescan be injected intradermally in a region of skin containing at least aportion of a tattoo to a depth from about 0.3 mm to about 1.25 mm,preferably about 1 mm from the skin's surface. Methods and devices forintradermal delivery of active agents are well known in the art.Illustrative methods for intradermal delivery of active agents toregions of skin can include direct intradermal (ID) administration. Insome embodiments, administration of the compositions of the presenttechnology to into regions of skin containing at least a portion of atattoo can be achieved using, for example, microneedle-based injectionand infusion systems, or any other means known to one skilled in the artto accurately target the intradermal compartment. Exemplary devicesinclude those disclosed in PCT International Application PublicationNos. WO 01/02178, published Jan. 10, 2002; and WO 02/02179, publishedJan. 10, 2002, U.S. Pat. No. 6,494,865, issued Dec. 17, 2002 and U.S.Pat. No. 6,569,143 issued May 27, 2003; and U.S. Publication No.2005/0163711 A1, published Jun. 28, 2005; and all of which areincorporated herein by reference in their entirety. Micro-cannula- andmicroneedle-based methodologies and devices are also described in U.S.Publication No. 2002-0095134, published Jul. 18, 2002, which isincorporated herein by reference in its entirety. Standard steel cannulacan also be used for intradermal delivery using devices and methods asdescribed in U.S. Pat. No. 6,494,865, issued Dec. 17, 2002, which isincorporated herein by reference in its entirety. These methods anddevices include the delivery of agents through narrow gauge (30 G ornarrower) “micro-cannula” with a limited depth of penetration (typicallyranging from 100 μm to 2 mm), as defined by the total length of thecannula or the total length of the cannula that is exposed beyond adepth-limiting hub feature.

In other embodiments, the bisphosphonate particles and compositionscomprising the bisphosphonate particles may be formulated for topicaladministration using the topical formulations described herein. Thetopical formulations containing the bisphosphonate particles may beapplied topically to the tattoo or portions thereof using an effectiveamount of the topical formulation as to cause at least some fading ofthe tattoo after one or more applications of the topical formulation asdescribed herein.

Dosing

The term “effective amount” denotes an amount of the bisphosphonateparticle containing composition, which is effective in achieving thedesired therapeutic result, namely at least inducing some fading of thetattoo. The particular amount of the bisphosphonate particle containingcomposition that constitutes an effective amount may depend, at least inpart, on one or more factors. Such factors include, but are not limitedto, the particular bisphosphonate or diphosphonate, acid, salt, ester,hydrate, polymorph, hemihydrate, solvate, or derivative thereof beingadministered, the state of the subject's immune system (e.g.,suppressed, compromised, stimulated); the route of administering thebisphosphonate particle containing composition; the age of the tattoo;the type of pigments contained within the tattoo; the skill and/orexperience of the person who applied the tattoo; the overall size of thetattoo; and the desired result (i.e., reduction or complete removal).Accordingly, it is not practical to set forth generally the amount thatconstitutes an effective amount of a bisphosphonate or diphosphonate,acid, salt, ester, hydrate, polymorph, hemihydrate, solvate, orderivative thereof. Those of ordinary skill in the art, however, canreadily determine the appropriate amount with due consideration of suchfactors.

In some embodiments, the methods of the present invention includeadministering sufficient bisphosphonate or diphosphonate, acid, salt,ester, hydrate, polymorph, hemihydrate, solvate, or derivative thereofto provide a dose of, for example, from about 100 ng/kg to about 50mg/kg to the subject, although in some embodiments the method may beperformed by administering the bisphosphonate or diphosphonate, acid,salt, ester, hydrate, polymorph, hemihydrate, solvate, or derivativethereof in a dose outside this range. In some of these embodiments, themethod includes administering sufficient bisphosphonate ordiphosphonate, acid, salt, ester, hydrate, polymorph, hemihydrate,solvate, or derivative thereof to provide a dose of from about 0.01mg/kg to about 100 mg/kg to the subject, for example, a dose of fromabout 0.1 mg/kg to about 50 mg/kg. The artisan, by routine typeexperimentation should have no substantial difficulties in determiningthe effective amount in each case.

A suitable formulation may contain, for example, about 0.001%, about0.002%, about 0:005%, about 0.01%, about 0.015%, about 0.02%, about0.025%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%,about 1%, about 2.5%, about 5%, about 7.5%, about 10%, about 15%, orabout 20% active bisphosphonate, or diphosphonate, acid, salt, ester,hydrate, polymorph, hemihydrate, solvate, or derivative thereof on a(v/v) basis, for example, one or more of alendronate, cimadronate,incadronate, clodronate, etidronates, risedronate, zoledronate,ibandronate, minodronate, pamidronate, piridronate, tiludronate,olpadronate, neridronate, YH529, EB 1053 ISA-13-1 and pharmaceuticallyacceptable salts, esters and mixtures thereof. In one particularembodiment, the composition includes about 5% of one or more ofalendronate, cimadronate, incadronate, clodronate, etidronates,risedronate, zoledronate, ibandronate, minodronate, pamidronate,piridronate, tiludronate, olpadronate, neridronate, YH529, EB 1053ISA-13-1 and pharmaceutically acceptable salts, esters and mixturesthereof. In some embodiments, the amount of bisphosphonate in thecomposition ranges from about 10 mg/mL to about 500 mg/mL on a weightper volume basis.

In some embodiments of the invention, the composition of the presenttechnology containing bisphosphonate particles may be administered, forexample, from a single dose to multiple doses administered once ormultiple times per day. In certain embodiments, the bisphosphonateparticles appropriately formulated, may be administered from about onceper week to about once per day. In one particular embodiment, the dosedcomposition containing bisphosphonate particles is administered once perday. In an alternative embodiment, the dosed composition containingbisphosphonate particles is administered once every other day. In someembodiments, the dosed composition is administered to a region of skincontaining at least a portion of a tattoo, at least once per week, or atleast twice per week, or at least once per month, or at least 5-10 timesper month or at least 1-10 times per 6 months.

EXAMPLES Example 1. Preparation of Tattoo Removal Compositions

The following methodology was used to make 5 mL of 10% lipid solution(weight:volume) for preparing a liposomal particle encapsulated with thebisphosphonate clodronate. In a round 100 mL bottom flask the followingwere combined: 0.5 grams of 95% soy phosphotidyl choline (sPC) (AvantiLipids: 441601); 0.09 grams of Span80 (Sigma-Aldrich: S6760). To thismixture, 3 mL of chloroform/methanol (2:1) is added. The sPC and Span80are dissolved in the chloroform/methanol mixture and evaporated undervacuum in rotary evaporator until uniform lipid film forms inside theflask. In a separate vessel, clodronate (i.e. selected bisphosphonate)(Sigma-Aldrich: D4434) is dissolved in 7% ethanol (EtOH) to make a 200mg/mL concentration solution. The lipid film in the round bottom flaskis hydrated with 10 mL of EtOH containing 200 mg/mL of clodronate. Theflask is flushed with nitrogen and then capped. The flask is thenrotated for 1 hr at room temperature (RT). The flask is then rested forat least 2 hrs (or overnight) at room temperature. The liposomes areformed by sonication of the formulation at 40 W for 20 min at 4° C. TheRuined liposomes containing clodronate are then extruded through 400 nmmembrane twenty times. Subsequently, the liposomal formulation is thenextruded through 200 nm membrane twenty times. The resultant extrudedliposomal formulation is spun in a swinging rotor centrifuge for 8 hrsat 50,000 RPM at 10° C. The lower clear layer (i.e. free clodronate in7% EtOH) is removed and reused for further liposomal preparation. Theupper layer is resuspended in 7% EtOH. The resultant bisphosphonateparticle containing formulation (clodronate liposomes) is then spun in aswinging rotor centrifuge for 1 hr at 24,000 RPM at 10° C. Again, theupper floating layer is removed and the pellet is resuspended in 5 mL of7% EtOH for a final concentration of 10% liposomes (weight:volume). Theclodronate liposomes are then aliquoted and flushed with nitrogen,capped, and sealed with Teflon tape. The clodronate liposomes are thenstored at 4° C. in dark.

Example 2. Apoptosis of Ink Containing Macrophages with ClodronateLiposomes In Vivo

Using an established model of tattooing in albino, hairless,immunocompetent (SKH1) mice, various formulations of clodronateencapsulated liposomes were tested. In brief, male SKH1 mice wereobtained from Charles River (Boston, Mass.) at 8 weeks of age. The micewere housed at the Carleton Animal Care Facility at Dalhousie Universityand acclimated in the animal room for 2 week prior to use. Alltreatments of the mice conform to Canadian Council on Animal Careguidelines. At 10 weeks of age, mice were anesthetized with isofluraneand tattooed midscapular with two 1 cm² half-solid and half-linedtattoos 2 cm apart on each side of the spine using a 14-pt long-taperedtattoo needle (AIMS Inc., Hornell, N.Y.) on a commercial tattoo machine(AIMS Inc. Hornell, N.Y.). Tattoos were left to heal for 1 month priorto initiating treatment with the formulation.

At 1 month, the skin was wiped with dH₂O and 25 μL offluorescently-labeled (DiO⁺) formulation was applied to a tattooed sideof the midscapular region. The formulation was rubbed evenly on the areaof skin using a nitrile rubber glove and then left to dry uncovered. Atdifferent time points, the animals were sacrificed by isofluranefollowed by cervical dislocation. Skin and draining lymph nodes wereharvested. Each skin segment and lymph node was divided in half: (1)fixed in 4% PFA overnight and transferred to Millonig's buffer untilsunk in 2:1 OCT:sucrose (20%) or (2) fixed in 10% neutral-bufferedformalin and paraffin embedded for general histology and apoptosislabeling.

Frozen skin were cut at 7 μm on a cryostat and stored at 4° C. untiluse. Frozen sections were stained for CD11b (AbD Serotec) followed by anAlexa Fluor® 555 Goat anti-rat IgG (H+L) (Invitrogen). Nuclei werelabeled with Hoechst (H1399, Invitrogen).

Paraffin sections were cut at Sum and processed according to themanufacturer's protocol for the Apoptag peroxidase apoptosis kit(Millipore). F4/80, TUNEL, fluorescently labeled clodronate containingliposomes, and Hoechst will be visualized using a Zeiss Axioplan II andimaged with an AxioCam HRC Color Camera (Carl Zeiss International,Toronto, ON). Co-localization of F4/80-Alexa Fluor® 647, TUNEL-PE, andfluorescently labeled clodronate containing liposomes-DiO appear white.Tattoo ink appears opaque, thus allowing visualization of F4/80,fluorescently labeled clodronate containing liposomes, TUNEL, and thetattoo pigment.

Sections of skin from the treated mice reveal uptake of clodronateliposomes in vivo after 24 hours and after 2 weeks. In FIG. 1A, thephotomicrograph illustrates the dermal layers marked with white arrowsto indicate tattoo ink taken up by macrophages as seen with lightmicroscopy, and as shown in FIG. 1B, the photomicrograph illustrates thedermal layers marked with white arrows to indicate tattoo ink andclodronate liposomes taken up by macrophages as seen with fluorescencemicroscopy. The liposomes injected have a fluorescent dye and areclearly seen in the dermal layers after 24 hours. These fluorescentlylabeled liposomes containing clodronate are taken up by residentmacrophages as indicated in FIG. 1C illustrating an overlay of the lightmicroscopy structures with the presence of macrophages havingphagocytosed the clodronate liposomes as seen with fluorescencemicroscopy.

It is believed that phagocytic cells such as macrophages take up inkparticles and proceed to transport the ink to the draining lymph nodevia the lymphatic system. The results shown in FIGS. 1D-F illustratethat at the first time point of 24 hours some of the ink has beentransported to the lymph nodes.

However, after 2 weeks, the macrophages containing the clodronateliposomes are visibly seen transporting ink to the lymph nodes as shownin FIGS. 1G-H.

To confirm that the macrophages which have taken up the ink and theliposomes are sensitive to the apoptotic activity of the liposomalpreparation administered, FIG. 2 depicts a photomicrograph representingdermal skin tissue in the mice model with clodronate liposomes of thepresent technology and an apoptosis antibody to illustrate the presenceof apoptotic macrophages. As can be seen in FIG. 2, apoptoticmacrophages are seen with digested tattoo ink particles. These resultsstrongly suggest that the clodronate liposomes are preferentially takenup by the dermal macrophages. The macrophages containing ink particlesalso take up the clodronate encapsulated liposomes and undergo apoptosiswhich results in a reduction in the amount of ink particles present inthe treated skin.

Example 3. Uptake of Foreign Ink Particles by Macrophages

The ability to induce macrophage cell death using liposomal clodronatein vitro has been demonstrated by the inventor's laboratory. An in-vivoprotocol for removing tattoos can be performed as described. Pigs (atleast 4 per group—4 groups, two treatment groups and two control groups)are tattooed by a professional tattoo artist, to best mimic aprofessionally administered tattoo on a human. The tattoo will bedivided into a representative number of regions ranging from 1 to 10. Abisphosphonate particle composition comprising liposomal clodronate in7% ethanol as described in Example 1 will be prepared. Clodronateencapsulated liposomes (test) and control liposomes (control) for twoforms of administration will be prepared: (1) intradermal (injection)and (2) transdermal (cream). The tattoos will be allowed to heal priorto beginning the administration of the control and test liposomes. Thecontrol and test liposomes are administered (100 μL of the liposomalpreparation intradermally or 100 mg of a cream containing 100 μL of theliposomal preparation—each administered dose containing a liposomalconcentration of 10 mg/kg weight of the subject) to one or more regionsof the tattooed skin at day 0, and then once daily for a period of tendays. Tattoos will be photographed each day, beginning at the initialadministration and ending with cessation of treatment and then fourweeks after the cessation of treatment. The photographs are taken underidentical light settings. The degree of fading is visually qualitativelymeasured using a +, ++, +++ and ++++ scale representing: +˜10% fading,++ representing ˜10-33% fading, +++ representing ˜34-66% fading and ++++representing >66% percent fading, each image compared to the tattooimage taken at day 0.

Example 4. Tattoo Ink Removal In Vivo

Male SKH1 mice were obtained from Charles River (Boston, Mass.) at 8weeks of age. The mice were housed at the Carleton Animal Care Facilityat Dalhousie University and acclimated in the animal room for 2 weekprior to use. All treatments of the mice conform to Canadian Council onAnimal Care guidelines. The animals were tattooed on the back with 4×4cm longitudinal lines using Phoenix TAT2 black ink, which were then leftto heal. After approximately 12 weeks of healing, the site of the tattoowas topically treated with a composition of the present invention. Thetopically administered treatment is a composition (25 microliters)comprised of a solution containing 33.07 mM lipid containingapproximately 3 mg/mL of clodronate in a medium of 7% ethanol at pH 7.2,(hereinafter “clodronate containing liposomal composition”). Each doseof the clodronate containing liposomal composition contained 25microliters of the clodronate containing liposomal composition wastopically applied and equally spread over a 4 cm line of tattoo. Thetattooed skin was treated with one dose per week, for a total of 12doses per treatment site. Sections of tattooed skin and lymph nodes fromtreated and non-treated sites were removed at the following time points:t=day 1 (FIGS. 4, 5, 9, and 11), t=day 2 (FIGS. 5, 6, and 11), t=day 5(FIGS. 5 and 11), t=day 7 (FIGS. 5, 7, 8, and 11), t=day 14 (FIGS. 5, 8,9, 10, and 11) and t=12 weeks (FIGS. 3, and 12). Complete skin sectionswere removed capturing the epidermis and dermal layers of skin andfurther analysed with light microscopy or fluorescence staining.

Mice were tattooed and left to heal for 3 months before initiatingtreatment. FIG. 3 depicts a representative paraffin section of a healedtattoo stained with hematoxylin (nuclei) and eosin (cytoplasm). In thehealed tattoo, the tattoo ink in the upper dermis has largely beenrelocated to the inner dermis. As shown in FIGS. 4A-4C, skin wastopically treated with fluorescently labeled clodronate containingliposomes. As shown in FIG. 4A, light microscopy photomicrograph of theskin section shows dispersion of the tattoo skin ink throughout thereticular dermis. FIG. 4B depicts fluorescent labeled clodronatecontaining liposomes. FIG. 4C depicts an overlay image of FIG. 4A andFIG. 4B of skin from an animal treated with fluorescently labeledclodronate containing liposomes and harvested 24 hrs later. Tattoopigment can be seen as opaque black. Unhealed tattoo ink can be seen inthe reticular dermis. Colocalization of tattoo ink and fluorescentlylabeled clodronate containing liposomes can be seen in multiple areas.Image was taken at 40×.

FIGS. 5A-5E represent fluorescent photomicrograph images of skin frommice treated with fluorescently labeled clodronate containing liposomesat t=day 0 and harvested shown in FIG. 5A at t=day 1, FIG. 5B at t=day2, FIG. 5C at t=day 5, FIG. 5D at t=day 7 and FIG. 5E at day 14. Tattoopigment can be seen as opaque black. Colocalization of tattoo ink andfluorescently labeled clodronate containing liposomes appears to peak at48 hrs. Images were taken at 40×.

As shown in FIGS. 6A and 6B representative (6A) skin and (6B) lymph nodesections from mice treated with clodronate containing liposomalcomposition at t=0, harvested at day 2, and stained for TUNEL(apoptosis). TUNEL was developed with DAB (brown). Tattoo pigment can beseen as opaque black. Colocalization of tattoo ink and apoptosis can beseen in multiple cells in the skin as shown in FIG. 6A. Pigment can beseen in the lymph node, but there is a marked absence of apoptosis inthe lymph node section as shown in FIG. 6B. Images were taken at 63×.

As shown in FIG. 7 a representative image of CD11b+ cell (macrophagesand monocytes) efflux from the skin to the lymph nodes following dosingwith fluorescently labeled clodronate containing liposomes at t=0 andharvesting at day 7. Tattoo ink appears as opaque.

In other experiments, mice were treated (or untreated) with liposomalcompositions containing liposomes embedded with clodronate as describedin the present examples. Mice were treated (topically) at one time witha dose of the clodronate containing liposomal preparation, and skin washarvested at either 1 week or 2 weeks post application. Skin sectionswere embedded in paraffin and cut at 5 microns. Slides were stained withhematoxylin (nuclei) and eosin (cytoplasm). FIGS. 8A-8D are orientedwith the epidermis at the bottom. In the treated sections shown in FIGS.8B and 8D, the tattoo ink has been mostly removed from the deeper dermallayer (mid to upper part of each photo) by 1 week and virtuallycompletely removed by 2 weeks post application. This indicates that theink that resides in the inner dermal layer (the “permanent” ink) isbeing removed in a gradual process dependent on bisphosphonate liposomaltattoo removal compositions of the present invention. As shown in FIGS.8A and 8C, permanent tattoo ink is trapped intra and extracellularly inthe inner and upper dermal layers.

In further experiments, lymph nodes from mice untreated and treated withfluorescently labeled liposomal compositions containing clodronate asdescribed in the present example were sectioned. As shown in FIGS. 9Aand 9D, lymph node sections taken after 1 day and 14 days respectivelywere imaged using light microscopy. In FIGS. 9B and 9E, sections of nodetissue were fluorescently stained to visualize fluorescently labeledclodronate containing particles and/or cell debris. In each of thetissue staining methods, the top row illustrates photomicrographs of asection of lymph node from an animal treated 24 hrs prior to and thesecond row reflects node tissue obtained after 14 days of treatment. Anoverlay showing the tattoo ink particles relative to the fluorescentlylabeled liposomal compositions containing clodronate are shown in FIGS.9C and 9F. FIG. 9G is shown as an enlargement of an image depicting 2 wklymph node in the white box. Colocalization of tattoo ink andfluorescently labeled clodronate containing liposomes/cell debris can beseen in multiple areas, indicated by white arrows, which appears toincrease with time after dosing with clodronate containing liposomes.Lymph nodes were negative for apoptosis markers, suggesting greenlabeling is due to cell debris and not active clodronate liposomemediated removal, and thus suggests, that clodronate containing liposomeremoval accelerates tattoo ink removal from the dermis to draining lymphnode, as indicated by the colocalization not tattoo ink and cellulardebris. Images were taken at 40×.

FIG. 10 depicts a photomicrograph of a lymph node from a representativemouse treated with fluorescently labeled clodronate containing liposomesand harvested 14 days after the initial dose. Monocyte/macrophage markerCD11b is distinguishable from other fluorescent staining and is seen tocolocalize with the fluorescently labeled clodronate containingliposomes/cell debris. Tattoo ink can be seen as opaque black.Colocalization of tattoo ink and fluorescently labeled clodronatecontaining liposomes/cell debris can be seen in multiple areas,indicated by white arrows. Lymph nodes were negative for apoptosismarkers, suggesting that the presence of green fluorescence labeling iscell debris and not active fluorescently labeled clodronate containingliposomes. Image was taken at 100×.

Representative photomicrographs are shown in FIGS. 11A-11E, depictingimages of lymph node sections from mice treated with fluorescentlylabeled clodronate containing liposomes at t=0 and harvested at day 1(FIG. 11A), at day 2 (FIG. 11B), at day 5 (FIG. 11C), at day 7 (FIG.11D), at day 14 (FIG. 11E). Tattoo ink can be seen as opaque black.Colocalization of tattoo ink and fluorescently labeled clodronatecontaining liposomes in the lymph nodes appears to peak at 5-14 days.Images were taken at 40×.

Mice were treated weekly with a clodronate containing liposomalpreparation, or liposomal control. Skin sections were subsequentlyharvested after 12 weeks of one time weekly dosing. Skin sections wereembedded in paraffin and cut at 5 microns. Slides were stained withhematoxylin (nuclei) and eosin (cytoplasm). As shown in FIG. 12B, theskin section illustrates removal of tattoo ink from the deeper dermallayer leaving extracellular tattoo ink which is invulnerable toclodronate liposomal based ink removal. In contrast, in therepresentative liposomal control section, as shown in FIG. 12 B, thetattoo ink remains in both the deeper dermal layer and the upper dermis.

Results and Conclusion:

The results obtained as shown in FIGS. 3-12 illustrate that theliposomal formulation was found to penetrate the skin after beingapplied topically, at the tattoo site into the deeper dermis, where itwas able to colocalize with areas of tattoo ink.

In addition, the clodronate containing liposomes, labeled with lipidsoluble fluorescent dye, were found to be removed from the skin by day7. While not wishing to be bound to any specific theory, it is believedthat the administration of the clodronate containing liposomalformulation to a tattoo covered skin, led to the targeting of cellswhich had taken up the tattoo ink for apoptosis in the deeper dermis,which was consistent with the pattern of macrophage infiltration, asdenoted by CD11b+ cells shown in FIG. 7. In the skin, the administrationof the clodronate containing liposomal formulation was associated with avisual reduction in the amount of tattoo ink present in the deeperdermis relative to the untreated control skin as shown in FIGS. 12A and12B. This difference was even more pronounced by 2 weeks post-treatment.Concurrently, while apoptosis was absent from the lymph nodes, there wasa marked increase in fluorescence between day one and day fourteenpost-treatment, consistent with removal of cells targeted in the skin bythe fluorescent liposome formulation. Moreover, in the lymph nodes,CD11b+ cells colocalized with tattoo ink and the green fluorescence,which together with the absence of apoptosis, supports one likelymechanism of action for the bisphosphonate containing liposomalformulation of the present invention. Finally, by 12 weeks of weeklytreatment, tattoo ink was virtually absent from the deeper dermis oftreated animals relative to untreated animals. The remaining tattoo inkin the skin was largely localized to the upper dermis and was found tobe extracellular.

What is claimed is:
 1. A topical, transdermal or intradermal compositionfor removing or fading a tattoo located on a region of skin on asubject, the composition comprising a formulation for application to theregion of the skin, wherein the formulation comprises liposomes composedof a free bisphosphonate, a liposome forming lipid, an emulsifier and alower alcohol diluent, wherein the ratio of liposome forming lipid toemulsifier ranges from 80:20 to 90:10.
 2. The composition of claim 1,wherein the bisphosphonate comprises: alendronate, cimadronate,incadronate, clodronate, etidronates, risedronate, zoledronate,ibandronate, minodronate, pamidronate, piridronate, tiludronate,olpadronate, neridronate, YH529, EB 1053 ISA-13-1 and pharmaceuticallyacceptable salts, esters and mixtures thereof.
 3. The composition ofclaim 2, wherein the bisphosphonate is clodronate or a pharmaceuticallyacceptable salt or ester thereof.
 4. The composition of claim 1, whereinthe liposome forming lipid is selected from the group consisting of soyphosphatidylcholine, hydrogenated soy phosphatidylcholine,distearoylphosphatidylcholine, sphingomyelin, diacylglycerol,phosphatidyl ethanolamine, phosphatidylglycerol,distearylphosphatidylcholine and distearylphosphatidyl ethanolamine andcombinations thereof.
 5. The composition of claim 4, wherein theliposome forming lipid is soy phosphatidylcholine.
 6. The composition ofclaim 1, wherein the emulsifier is a non-ionic surfactant.
 7. Thecomposition of claim 6, wherein the non-ionic surfactant is sorbitanmonooleate.
 8. The composition of claim 2, wherein the bisphosphonate isalendronate or a pharmaceutically acceptable salt or ester thereof. 9.The composition of claim 2, wherein the bisphosphonate is risedronate ora pharmaceutically acceptable salt or ester thereof.
 10. The compositionof claim 2, wherein the bisphosphonate is zoledronate or apharmaceutically acceptable salt or ester thereof.
 11. The compositionof claim 2, wherein the bisphosphonate is ibandronate or apharmaceutically acceptable salt or ester thereof.
 12. The compositionof claim 2, wherein the bisphosphonate is pamidronate or apharmaceutically acceptable salt or ester thereof.
 13. The compositionof claim 1, wherein the composition is a topical formulation comprisinga cream, an emulsion, a foam, an ointment, a dispersion, a paste, aspray, a solution, an oil, or a microemulsion.
 14. The composition ofclaim 1, wherein the composition is a transdermal formulation comprisinga transdermal patch.
 15. The composition of claim 1, wherein thecomposition is an intradermal formulation comprising a solution,emulsion, suspension or dispersion.